Polypeptide with growth hormone receptor agonist properties, nucleic acid coding said polypeptide, expression vector thereof and cell producing said polypeptide

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to genetic and protein engineering and can be used in biomedical industry. A genetic make up is proposed, which codes a peptide in which two domains bonding the growth hormone (GH) receptor are bonded into a tandem by a "semirigid" or "rigid" linker, which consists of at least 1-4 copies of the A(EAAAK)A amino acid sequence.

EFFECT: as a result of expression of the nucleotide sequence coding the said tandem, GH-linker-GH polypeptides are obtained, which exhibit growth hormone receptor agonist properties, which determine the possibility of their use in medicinal agents for treating diseases related to the need to administer the growth hormone.

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This invention relates to polypeptides that contain at least two domains which are able to bind to the receptor of the cytokine, where these domains are connected by a peptide linker molecule.

The group of growth factors called cytokines, is involved in a number of different cellular functions. These functions include, as an example, but not limitation, modulation of the immune system, regulation of energy metabolism and the control of growth and development. Cytokines mediate their effects through receptors expressed on the cell surface of target cells. Receptors of cytokines can be divided into three separate subgroups. The receptor type 1 (family of growth hormone (GH)are characterized by four conservative cysteine residues in aminobenzene part of their extracellular domain and the presence of the conservative motif Trp-Sex-Xaa-Trp-Ser in the C-terminal part. Duplicate Cys motif is also present in type 2 (family of interferon) and type 3 (the family of tumor necrosis factor).

It is known that many domains of cytokines interact with their recognition through specific receptor sites. Some receptors cytokines have both binding sites domain with high affinity, and the binding sites with low affinity.

For example, it is known that one molecule of GH is associated with two molecules Rotz the Torah (GHR) (Cunningham et al., 1991; de Vos et al., 1992; Sundstrom et al., 1996; Clackson et al., 1998). This is done through two unique receptor-binding site on GH and General pocket binding to the extracellular domain of the two receptors. Site 1 on the GH molecule has a higher affinity than site 2, and suppose that the dimerization of the receptor occurs sequentially, with one receptor binds to site 1 on GH, with subsequent recruitment of the second receptor site 2. The extracellular domain of the GHR exists in the form of two related domains, each of which consists of approximately 100 amino acids. It is a conformational change in these two domains occurs when the binding of the hormone with the formation of the trimeric complex GHR-GH-GHR. After internalization of the complex GHR-GH-GHR should stage recycling, whereby the receptor molecule is regenerated for further use in the cell.

Cytokines and other domains often form complexes receptor domain when linking. The receptors involved in the formation of complexes can be homogeneous or heterogeneous. For example, erythropoietin and GH form a trimeric complex of the receptor-hormone receptor. Interleukin-4 forms a trimeric complex with the receptor-hormone-another receptor. The tumor necrosis factor induces signal through education homotypic of trimers cellular transmembrane receptor necrosis factor op is Halsey, TNF-1/p55 or TNF-2/p75. Other cytokines, such as leptin and GCSF (granulocyte colony-stimulating factor), form a tetramer complexes of receptor-hormone-hormone receptor, and some cytokines (such as interleukin 6) may form a review of the complexes consisting of two soluble receptor molecules, two transmembrane receptor molecules and two molecules of the cytokine. In each case there is a primary binding site with high affinity, which localizes the cytokine in complex with the receptor, and additional sites that play a secondary role in changing the conformation or the recruitment of other molecules, initiating, by this means, the signal transmission.

Superfamily of cytokines TNF (tumour necrosis factor, tumor necrosis factor) activates transmission of a signal for survival, death and differentiation, which regulate the development, organization and homeostasis of lymphoid tissue, breast tissue, neural and ectodermal tissues. For TNF demonstrated its role in protecting the host, which includes, for example, differentiation of cells of the spleen, full lgG response and isotype switching, activation of macrophages, the generation of nitric oxide and radicals of reactive oxygen species. However, overexpression of TNF is also involved in the pathogenesis. Proof of such participation found in the following pathologies: the tank is arianny sepsis; graft-versus-host; cerebral malaria; rheumatoid arthritis; alopecia areata / total baldness; asthma; cancer; Crohn's disease; diabetes; obesity; psoriasis and psoriatic arthritis, sarcoidosis, scleroderma, and toxic shock syndrome. These pathologies is considered established and/or potential pathologies for anti-TNF agents.

Overexpression of cytokines is the cause of several human diseases such as acromegaly, gigantism, GH deficiency, Turner syndrome, renal failure, osteoporosis, osteoarthritis, diabetes, cancer, obesity, insulin resistance, hyperlipidemia, hypertension, anemia, autoimmune and infectious diseases, inflammatory disorders, including rheumatoid arthritis.

Approach for inhibiting the action of cytokines, such as GH, prolactin, or TNF, is the introduction of the antagonists.

One example of a GH antagonist is pegvisomant, which is a modified molecule GH, covered with polyethylene glycol (PEG). Pegvisomant has several beneficial effects, including, for example, reduced glomerular filtration rate, due to increased effective molecular weight, which reduces the dose required for obtaining the desired effect [see Abuchowski et al J Biol Chem., 252, 3578-3581, (1977)]. However, sredstv who eat paglierani is reduced affinity of the molecules of the modified GH to GHR.

An example of a prolactin antagonist disclosed in WO 03/057729 (which are included in this description of the invention in its entirety by reference, and, more specifically, protein sequence and the nucleotide sequence encoding the specified prolactin antagonist). The prolactin antagonist contains a modified amino acid sequence of human prolactin, which is a replacement of the glycine residue at position 129 of the arginine residue. The modified protein prolactin acts as an inhibitor of activation of the prolactin receptor.

Has developed a number of therapeutic strategies for inhibiting TNF-based opportunities: 1) to inhibit the synthesis of TNF (e.g., using the inhibitory cytokines IL-10; thalidomide, corticosteroids, cyclosporine A; antisense oligonucleotides); 2) to inhibit the processing of TNF (e.g., inhibitors of metalloprotease (TACE, TNF-α converting enzyme)); 3) to neutralize TNF (e.g., using soluble TNF receptors or antibodies to TNF).

The authors of the present invention describe polypeptides containing multiple ligand-binding domains of cytokine receptors and their use in the modulation of receptor-mediated activation of cytokines.

According to one aspect of the present invention proposed a polypeptide containing at least two associated cytokine is their domain, the ability to communicate with cytokine receptor, where these domains are connected by a peptide linker molecule, which contains the rigid spiral section.

In the preferred embodiment of the present invention the specified polypeptide acts as an antagonist of the specified(s) cytokine(s) receptor(s). Alternatively, the specified polypeptide acts as an agonist.

Preferably, the polypeptide contains domains that are located in tandem order. In preferred embodiments of the invention the polypeptide contains 2, 3, 4, 5, 6, 7, 8, 9 or 10 domains in tandem order.

In still another preferred embodiment of the invention the polypeptide contains more than 10 domains in tandem order.

Preferably rigid spiral section contains at least one copy of the motif AND(EAAAK)xAnd or its functional variant. Preferably, the peptide linker molecule contains two copies of the motif EAAAK, and the length of the molecules of a given peptide linker can be increased by the step of adding at least one amino acid.

"Functional variant" is a linker molecule, which may differ in amino acid sequence of one or more than one substitution, insertion, deletion, but which essentially retains the spiral or aspirin the Yu conformation. Among the preferred options are options that differ from the reference amino acid sequence are conservative amino acid substitutions. Such substitutions are substitutions in which the amino acid is replaced with another amino acid with similar characteristics. The following non-limiting list of amino acids regarded as conservative substitutions (similar): (a) alanine, serine and threonine; b) glutamic acid and aspartic acid; asparagine and glutamine; arginine, lysine and histidine; d) isoleucine, leucine, methionine and valine and (e) phenylalanine, tyrosine and tryptophan. Most preferred are amino acid substitutions, which essentially remain flexible or rigid helical linker section.

In another preferred embodiment of the present invention the linker molecule comprises at least one flexible non-spiral plot.

While the presence of the rigid spiral section supports spatial separation of domains, as described above, the flexible nonhelical plot allows domains to be oriented in the binding sites of cytokine(s) receptor(s).

In one embodiment of the present invention flexible nonhelical the site is located on aminoterminal the end of the molecule peptide linker molecules or about him, who eat the most making possible the orientation of the binding domain, located on aminoterminal the end of the peptide linker molecule, relative to its recognition receptor.

In another embodiment of the present invention flexible nonhelical the site is located on carboxyterminal the end of the peptide linker molecule or around it, thereby making possible the orientation of the binding domain, located on carboxyterminal the end of the peptide linker molecule, relative to its recognition receptor.

In one another embodiment of the present invention flexible nonhelical the site is located at the amino - and carboxyterminal the end of the peptide linker molecule or around them, thereby making possible the orientation of the binding domains located at the amino - and carboxyterminal the end of the peptide linker molecules with respect to their recognition receptors.

Preferably flexible nonhelical the site is located near at least one binding domain. Even more preferably flexible nonhelical plot forms a connection between the binding domain and rigid spiral section.

Even more preferably rigid spiral section contains at least one copy of the motif AND(EAAAK)xA. the length of the rigid nonhelical region can be increased by increasing the number of repetitions of this mod is willow AND(EAAAK) xA.

In the preferred embodiment of the present invention "x" motif a(EAAAK)xAnd is less than 10 copies. Even more preferably, x is less than 5 copies. Even more preferably, x is selected from 1, 2, 3, 4 or 5 copies.

In the preferred embodiment of the present invention is not flexible connections between rigid alpha-helical linkers and linking domains, but these binding domains are directly linked rigid alpha-helical linker.

In the preferred embodiment of this invention these binding domains are connected by a connecting molecule, consisting of a rigid alpha-helix.

In the preferred embodiment of the present invention specified helical linker molecule connects the carboxyl end of one binding domain aminocom.com second binding domain.

In this embodiment of the present invention the helical linker is continuous between the C-terminal helix molecules of the first cytokine and N-terminal helix molecules of the second cytokine, hard binding, thus, the two cytokine-binding domain in a substantially fixed orientation. For example, this may include deletion of the short N-terminal and post-helical C-terminal segment of the first domain of the cytokine and short preserving N-terminal part is ka from the second cytokine (for example, residues 182 to 190 of the first cytokine and residues 1-5 of a second cytokine, as they are short sections of random ring conformation after the C-terminal helix (for example, helix 4 Figb) in the first cytokine and before the N-end (spiral 1' Figb) second cytokine.

In various constructs that fixed orientation (both translational and rotational) can be changed either by inserting 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids, or by deletions 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in order to obtain molecules with new properties, for example with antagonistic properties. Adding additional amino acids will give additional relative movement of the two domains of approximately 1.5 Å and the relative rotation of the two domains around the helical axis by approximately +100°. Typical linkers can start with two units EAAAK and will be lengthened by adding sequences A, AA, AAA, AAAA, AAAAA and EAAC.

In another preferred embodiment of the present invention binding domains of the polypeptide is identical or similar to each other.

In addition, in other embodiments of the present invention, the polypeptide contains binding domains of the cytokines selected from the group consisting of growth hormone, leptin, erythropoietin, prolactin, interleukin (IL) IL-2, IL-3, IL-4, IL-5, IL-6, IL-, IL-9, IL-10, IL-11, R35 subunit of IL-12, IL-13, IL-15; granulocyte colony-stimulating factor (G-CSF); granulocyte-macrophage colony-stimulating factor (GM-CSF); ciliary neurotrophic factor (CNTF); cardiotrophin (ST-1); factor ingibirovalo leukocyte migration (LIF); oncostatin M (OSM); interferon, IFNα and IFNγ, tumor necrosis factor (TNF)α, TNFβ and RANK ligand.

In another preferred embodiment of the present invention at least one domain contains the binding domain of growth hormone.

In another preferred embodiment of the present invention the specified polypeptide contains at least two binding domain of growth hormone or a variant of growth hormone.

Modified GH variants are disclosed in US 5849535, which are included in this description by reference. Modification of GH are both in binding site 1 and site 2. Modifications to the website produce 1 molecule of GH, which has a higher affinity for GHR compared with wild-type GH. These modified molecules GH act as agonists. There is also the disclosure of modifications of site 2, which lead to the creation of GH antagonists. Additional examples of modifications GH for site 1, which change the affinity of binding of GH are disclosed in US 5854026; US 6004931; US 6022711; US 6057292 and US 6136563, each of which are included in this description of the invention in which redstem links. Also described modification of site 2, in particular amino acid residue G120 in GH, which when modifying in any of arginine, lysine, tryptophan, tyrosine, phenylalanine or glutamic acid create a GH molecule with the antagonist properties.

Located on the simultaneous consideration of the patent application WO 03/070765 authors of the present invention, which is incorporated in this description by reference, the inventors describe the product of the merger of the variant GH with antagonistic activity against activation of the GH receptor. This option GH fused via a flexible linker with the extracellular domain of the receptor for growth hormone. This chimeric polypeptide demonstrates slow clearance and antagonistic activity. The proposal is similar chimeric polypeptide, but with rigid or partially flexible linker is also within the scope of the invention disclosed here.

In an alternative preferred embodiment of the present invention the specified polypeptide contains at least two binding domain of the prolactin or variant of prolactin.

In the preferred embodiment of the present invention the specified polypeptide variants of prolactin contains an amino acid sequence that is modified at position 129 of the human prolactin.

In the preferred embodiment of this is bretania this modification is an amino acid replacement. Preferably the specified substitution is a substitution of amino acid residue glycine residue in the amino acid arginine. Preferably this modification further comprises a deletion of at least 9, 10, 11, 12, 13 or 14 aminobenzene amino acid residues.

In an alternate embodiment of the present invention binding domains of the polypeptide differ from each other.

In the preferred embodiment of the present invention the specified polypeptide contains a first binding domain, which is a binding domain of growth hormone, and a second binding domain, which represents the binding domain of the prolactin.

Preferably the specified polypeptide comprises the binding domain of growth hormone and binding domain of the prolactin.

In an alternative preferred embodiment of the present invention the specified polypeptide contains a first binding domain, which is a modified binding domain of growth hormone, and a second binding domain, which is a modified binding domain of the prolactin.

Preferably the specified polypeptide comprises a modified binding domain of growth hormone and modified binding domain of the prolactin.

In the preferred embodiment of the present invention, the specified option is qualified binding domain of growth hormone contains the amino acid substitution at position amino acids glycine 120. Preferably this modification is a substitution of glycine 120 amino acid selected from the group consisting of arginine, lysine, tryptophan, tyrosine, phenylalanine or glutamic acid.

In the preferred embodiment of this invention, this modification is a substitution of glycine 120 the amino acid residue arginine.

In an additional preferred embodiment of the present invention specified modified binding domain of the prolactin contains modification of glycine 129. Preferably this modification is a substitution of glycine 129 the amino acid residue arginine. Preferably this modification further comprises a deletion of at least 9, 10, 11, 12, 13 or 14 aminobenzene amino acid residues.

In an additional preferred embodiment of the present invention the specified polypeptide further comprises a ligand-binding domain of a cytokine receptor. Preferably the specified receptor is a receptor for growth hormone. In an alternative preferred embodiment of the present invention specified receptor is a receptor for prolactin.

In the preferred embodiment of the present invention, the specified ligand-binding domain can be connected with the specified binding domain of the cytokine linker, sod is Rasim rigid spiral section or consisting of him.

According to another aspect of the present invention proposed a nucleic acid molecule which encodes a polypeptide according to this invention.

In the preferred embodiment of the present invention, a specified nucleic acid molecule is a vector adapted for expression of the specified polypeptide.

Typically this adaptation includes providing sequences control transcription (promoter sequences), which mediate the expression of specific cells/tissues. These promoter sequences can be specific for cells/tissues, induced or constitutive.

The promoter is recognized in the field by the term and, for the sake of clarity, includes the following features, which are given only as an example. Enhancer elements are CIS-acting nucleic acid sequences which are often located 5' relative to the site of initiation of transcription of the gene (enhancers can also be found 3' to the gene sequence, or can even be localized in intron sequences and, therefore, are independent of position). The function of enhancers is to increase the rate of transcription of the gene is associated with enhancer. The activity enhancer is sensitive is th to TRANS-acting transcription factors (polypeptides), which has been shown to be specifically associated with enhancer elements. The binding/activity of transcription factors (please refer to the Eukaryotic reduced Factors, by David S Latchman, Academic Press Ltd, San Diego) are sensitive to a variety of environmental stimuli, which include, for example, intermediate metabolites (e.g. glucose), the effectors of the environment (e.g., heat).

Promoter elements also include the so-called TATA box and selection sequence initiation RNA polymerase (RIS), the function of which is to select the site of transcription initiation. These sequences also bind polypeptides, whose function, among others, is to facilitate the choice of transcription initiation RNA polymerase.

Adaptation also include the provision of breeding markers and sequences of Autonomous replication, which facilitates the maintenance of the vector in a eukaryotic or prokaryotic cell. Vectors that are supported offline, referred to as episomal vectors.

Adaptations that facilitate the expression of genes encoded by the vector, include the sequences of transcription termination/polyadenylation, which also includes ensuring internal binding sites of the ribosome (IRES), the function of which is to maximize the expression of genes encoded by ve the torus, organized in bicistronic or multicyclone cassette expression.

These adaptations are well known in this field. There is a significant amount of published literature regarding the construction of expression vectors and recombinant DNA technology in General. Please see Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, NY, and references in this publication; Marston, F (1987) DNA Cloning Techniques: A Practical Approach, Vol III, IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

For professionals in this field will be obvious that the vectors according to the invention can be vectors for gene therapy. Vectors for gene therapy are usually based on the viruses. A number of viruses are commonly used as vectors for the delivery of exogenous genes. Commonly used vectors include recombinante modified DNA and RNA viruses with shell or without shell, preferably selected from baculoviridiae (baculoviruses), parvoviridiae (parvoviruses), picornoviridiae (picornavirus), herpesviridiae (herpes viruses), poxviridiae (poxviruses), adenoviridiae (adenoviruses) or picornaviridiae (picornaviruses). You can also use chimeric vectors that exploit the useful elements of each property of the parent vector (see, for example, Feng, et al. (1997) Nature Biotechnology 15:866-870). Such viral vectors may the be vectors of wild-type or can be modified by the methods of recombinant DNA, to be deficient in replication can replicate conditionally or competent for replication.

Preferred vectors originate from adenovirus associated with adenoviral and retroviral genomes. In the preferred implementation of this invention, the vectors originate from the genome of a human adenovirus. Particularly preferred vectors originate from serotypes 2 or 5 human adenovirus. The ability to replicate such vectors can be reduced (to the point that is considered "deficient in replication") by modifications or deletions in the coding regions EA and/or 1b. Preferred are other modifications of the viral genome to achieve specific peculiarities of expression or provide re-introduction, or less immune response.

Alternatively, viral vectors can be conditionally can replicate or replication competent. Conventionally can replicate viral vectors are used to achieve selective expression in specific cell types, while avoiding unwanted infection broad spectrum. Examples of conventionally can replicate vectors described in Pennisi, E. (1996) Science 274:342-343; Russell, and S.J. (1994) Eur. J. of Cancer 30A(8):1165-1171. Additional examples can replicate selectively vectors include vectors, where a gene essential for replication Viru is a, is under the control of a promoter that is active only in a specific cell type or in a particular state of the cells so that in the absence of expression of this gene, the virus will not replicate. Examples of such vectors are described in Henderson et al., patent of the United States No. 5698443, issued December 16, 1997, and Henderson et al., patent of the United States No. 5871726, issued February 16, 1999, all ideas are included in this description by reference.

Additionally, the viral genome can be modified by including inducible promoters that allow for replication or expression only under certain conditions. Examples of inducible promoters are known in the scientific literature (see, e.g., Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; Iida, et al. (1996) J. Virol. 70(9):6054-6059; Hwang, et al. (1997) j Virol 71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al. (1997) J. Biol. Chem 272(46); 29364-29371.

Vectors can also be non-viral and is available from several commercial sources, easily accessible to the person skilled in the art. For example, the data vectors can be plasmids, which can be episomal or integrating.

According to another aspect of the present invention proposed a cell transformed or transfusiona nucleic acid or vector according to the invention.

In the preferred embodiment of the present invention the specified cell is a eukaryotic cell.

Preferably specified eukaryotic cell is selected from the group consisting of cells of fungi, such as Saccharomyces cerevisiae, Pichia spp; myxomycetes (e.g. Dictyostelium spp.); insect cells (for example Spodoptera frugiperda); plant cells, or mammalian cells (such as cell Cho (Chinese hamster ovary)).

In an alternative preferred embodiment of the present invention the specified cell is a prokaryotic cell.

In another aspect of the present invention, a method for obtaining a polypeptide according to this invention, which includes stages:

1) growing cells according to this invention under conditions leading to the production of the polypeptide according to the invention, and

2) release of the polypeptide from the cells or from its growth environment. In the preferred method according to this invention the specified polypeptide supplied affinity tag.

Affinity tags are known in the field and include binding protein maltose, glutathione-S-transferase, protein, calmodulin binding, and obtained by means of genetic engineering polyhistidine sites in proteins, which are then purified by affinity purification on matrices containing Nickel. In many cases, the stories in the sale of vectors and/or kits can be used to merge the protein of interest with a suitable affinity tag, with its subsequent transfection into the cell host for expression and subsequent extraction and purification on affinity matrix.

Located on the simultaneous consideration of the application WO 03/034275 authors of the present invention, the content of which is included in the description of this invention by reference, the inventors describe a new affinity tag for polypeptides, which uses domain comprising a signal sequence that directs the addition of glycosylphosphatidylinositol to the polypeptide. Polypeptides that include glycosylphosphatidylinositol label, preferably embedded in the lipid membrane and may have an antagonistic effect on the activation of receptors of cytokines. Therefore, the invention disclosed here encompasses polypeptides with attached molecule glycosylphosphatidylinositol.

According to another aspect of the present invention proposed a polypeptide containing the first cytokine binding domain connected to a second cytokine binding domain, where the specified polypeptide further comprises the extracellular domain of the cytokine receptor.

In the preferred embodiment of this invention, these first and second binding domains connected by a flexible linker molecule.

In an alternative preferred embodiment of the data is the first invention mentioned first and second binding domains are connected by a peptide linker molecule, which contains the rigid spiral section.

In the preferred embodiment of this invention, these first and second binding domains are connected by a peptide linker molecule containing rigid spiral section and a flexible non-spiral plot.

Peptide linkers that contain a rigid helical regions and combinations of rigid helical sections and flexible nonhelical plots were described above and are applicable to this embodiment of the present invention, as previously defined cytokines and cytokine receptors.

In the preferred embodiment of the present invention extracellular domain of the cytokine receptor is connected with the first or second cytokine binding domain via a linker molecule. Preferably the specified linker molecule contains rigid spiral section.

In an alternative preferred embodiment of the present invention specified linker molecule is flexible.

Preferably the specified linker molecule contains rigid spiral section and a flexible non-spiral plot.

In the preferred embodiment of the present invention indicated cytokine binding domain is a growth hormone or growth hormone and the extracellular domain is the extracellular domain of the growth hormone. Site is preferably specified domains are human.

The polypeptide according to this invention can exhibit dual functionality. First, the first and second domains, containing cytokines or parts thereof, which are preferably connected by a peptide linker molecule containing rigid spiral section which are able to bind with the receptors of cytokines, cell-surface and sterically hinder the Association of these receptors in receptor complexes, preventing, thus, subsequent cellular signal transmission. Secondly, the provision of the third domain containing receptors cytokines or their parts, provides the ability of functioning as a soluble receptor, binding, thus, any cytokine before binding with the receptor to the cell surface. This third domain is preferably connected with the first or with the second domain of the peptide linker molecule containing rigid spiral section. In an alternate embodiment of the present invention, the third domain is preferably connected to the first or second domain peptide linker molecule containing a flexible non-spiral plot.

In the preferred embodiment of the present invention indicated peptide linker molecules additionally contain an amino acid sequence that is sensitive to proteolytic cleavage.

According to the SNO another aspect of the present invention proposed the use of molecules of the polypeptide or nucleic acid according to the invention as pharmaceutical agents.

Preferably the proposed pharmaceutical composition comprising a molecule of the polypeptide or nucleic acid according to this invention. Preferably specified pharmaceutical composition comprises a carrier, excipient and/or diluent.

With the introduction of therapeutic compositions of the present invention is administered in pharmaceutically acceptable preparations. The term "pharmaceutically acceptable" means a non-toxic substance that has no harmful impact on the effectiveness of the biological activity of the active ingredients. These drugs are traditionally may contain salts, buffering agents, compatible carriers, preservatives and possibly other therapeutic agents.

When used in medicine, the salts should be pharmaceutically acceptable, however pharmaceutically unacceptable salts can be used to obtain pharmaceutically acceptable salts, and they are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include salts derived from the following acids: hydrochloric, Hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like, but are not limited to. Also pharmaceutically acceptable salts can be obtained in the form of alkali salts is full-time and alkaline earth metals, as, for example, salts of sodium, potassium or calcium.

Data pharmaceutical compositions may contain suitable buffering agents, including acetic acid in a salt; citric acid in a salt; boric acid salt and phosphoric acid salt.

Song data can be combined, if desired, with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier", as used here, means one or more than one compatible solid or liquid filler, diluent or encapsulating substance that is suitable for administration to humans. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the combined active ingredient for ease of use. The components of the pharmaceutical compositions can also be mixed together with molecules of the present invention and with each other so that there is no interaction, which essentially would impair the desired pharmaceutical efficiency.

Pharmaceutical compositions with convenience can be presented in a standard dosage form and may be obtained by any method well known in the field of pharmacy. All methods include the stage of bringing the active agent into Association with a carrier which constitutes one and the and more than one auxiliary ingredient. In General, these compositions are obtained by bringing the active compound in a homogeneous and close Association with a liquid carrier, a finely ground solid carriers or both and then, if necessary, by forming this product.

The data of the pharmaceutical compositions also may contain suitable preservatives, such as benzalkonium chloride; chlorobutanol; parabens and thimerosal.

The pharmaceutical compositions according to this invention can be entered by any conventional route, including injection or by gradual infusion over a certain period of time. The introduction may be, for example, oral, intravenous, intraperitoneal, intramuscular, intracavitary, subcutaneous or percutaneous.

Compositions suitable for oral administration may be presented as discrete units such as capsules, tablets, pellets, each of which contains a given number of active connections. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, an elixir, or an emulsion.

The composition of this invention is administered in effective amounts. "Effective amount" is a quantity of a composition which by itself or together with further doses it is desirable reacts the Y. In the case of treating a particular disease, such as cancer, the desired response is suppression of disease development. This may include only a temporary slowing of disease progression, although more preferably, it includes a permanent stop disease progression. This can be monitored by traditional methods.

Such amount will, of course, depend on the specific condition that is being treated, the severity of the condition, the individual parameters of the patient, including age, physical condition, size and weight, the duration of treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and competence of the practitioner. These factors are well known to specialists in this field and can be studied using no more than ordinary experimentation. Is usually preferable to use the maximum dose of the individual components or their combinations, that is, the highest safe dose according to sound medical decision. The usual experts in this field, however, it will be clear that the patient may insist on a lower dose or tolerable dose for medical reasons, psychological reasons, and, in fact, for any other reason.

In another and is the aspect of the present invention proposed the use of molecules of the polypeptide or nucleic acid according to the invention for the manufacture of a medicinal product for the treatment of diseases, selected from the group consisting of acromegaly, gigantism; GH deficiency, Turner syndrome; renal failure; osteoporosis; osteoarthritis; diabetes; cancer (such as prostate cancer, cervical cancer, breast cancer, melanoma, hepatoma, kidney cancer, glioma, bladder cancer, lung cancer, neural cancer, ovarian cancer, testicular cancer, pancreatic cancer, cancer of the gastrointestinal tract, lymphoma); obesity; insulin resistance; hyperlipidemia; hypertension; anemia; autoimmune and infectious diseases; inflammatory disorders, including rheumatoid arthritis.

According to this invention is also a method of treatment of the human or animal subject, comprising introducing an effective amount of the polypeptide, nucleic acid molecules, pharmaceutical compositions or treatments of this subject.

Throughout the description and claims the word "comprise" and "contain" and variations of these words, such as "include" and "includes"mean "including but not limited to", and they are not intended to exclude (and does not exclude other groups, additives, components, integers or stage.

Throughout the description and the claims the singular encompasses the plural unless the context otherwise requires. Specifically, when the use singular this description should be understood as considering the multiplicity and singularity, unless the context requires otherwise.

Properties, integers, characteristics, compounds, chemical group or groups described in connection with a particular aspect, embodiment or example of the present invention, should be understood as applicable to any other aspect, embodiment or example described herein unless it is incompatible with the given description.

Embodiments of the present invention will now be described only as an example and with reference to the following figures.

Figa. Cytokine domains (ovals) are connected alpha helix (dashed rectangle). Flexible linkers (curved arrows) connecting the first cytokine domain of the spiral and the spiral with a second cytokine domain; Figb. Cytokine domains are connected by an alpha helix. Flexible linkers connecting the first cytokine domain with the helical linker and helical linker to the second cytokine domain.

Figure 2. Helical linker has no flexible connectors - instead, it continues C-terminal helix (4) cytokine 1 and connects it to the N-terminal helix (1') cytokine 2 with the formation of rigid tandem connected to a single long spiral: 4 - linker helix 1'. The relative orientation of the two cytokine domains, following vetelino, is fixed. However, by creating different constructs using the attach or remove amino acids from the linker can generate a series of rigid tandems in which these domains are differently oriented.

Figure 3 illustrates the map and the sequence of nucleotides/amino acids to construct χ1C1b.

Figure 4 illustrates an overview of the structures of the linkers and primers used to generate tandem with the helical linkers with flexible ends.

Figure 5 illustrates the construction of border lands between the GH domains and linker to allow ligation of the primer duplexes with obtaining uninterrupted helical linkers between domains; B) primers used for modification χ1C1b obtaining χ15.

6 illustrates a map of the construct χ15 and sequence of the linker section.

7 illustrates an overview of the structure of the linker and the primers used to generate tandem with a rigid helical linkers.

Fig illustrates a schematic diagram showing the strategy design χ1L1.

Fig.9 illustrates the nucleotide sequence χ1L1. The GH domains are shown in gray, the domain of the GHR bold and linkers are underlined.

Figure 10 illustrates the amino acid sequence χ1L1. The GH domains are shown in gray, the domain of the GHR is edelen bold, and linkers are underlined.

11 illustrates a schematic diagram showing the cloning strategy for constructing χ1L1.

Fig illustrates the expression of χ1L1.

Fig illustrates the pre-cleaning χ1L1-His. χ1L1-His was purified using With2+-column.

Fig illustrates that χ1L1 shows agonistic activity.

Fig summarizes the nomenclature used in relation to rigid and semi-rigid constructs GH.

Fig: (a) is nukleinovokisly sequence of tandem GH containing a semi-rigid linker sequence; b) represents the amino acid sequence of the tandem GH containing a semi-rigid linker sequence; C) illustrates examples of a semi-rigid linkers used in the construction of tandems GH; g) illustrates a bacterial expression tandems GH containing semi-rigid linkers; and d) illustrates the bioactivity of tandems GH containing semi-rigid linkers.

Fig: (a) is nukleinovokisly sequence of tandem GH containing rigid linker sequence; b) represents the amino acid sequence of the tandem GH containing rigid linker sequence; C) illustrates examples of rigid linkers used in the construction of tandems GH; g) ill Trichet bacterial expression tandems GH, containing rigid linkers; and d) illustrates the bioactivity of tandems GH containing rigid linkers.

Figa illustrates the cleaning T1cEAK2+3his and analysis by staining Kumasi and Western blotting; Figb and 18B illustrate the bioactivity T1cEAK2+3his; Figg illustrates the cleaning T1cEAK2+4his and analysis by staining Kumasi and Western blotting; and Figd and 18E illustrate the bioactivity T1cEAK2+4his.

Fig illustrates ELISA for determination of tandem growth hormone.

Fig is a schematic illustration of tandem growth hormone associated flexible, semi-rigid and rigid linkers.

Fig illustrates examples of possible combinations of prolactin (PRL), growth hormone (GH) and their antagonistic mutants.

Fig illustrates nucleotide and amino acid sequences of prolactin and one of its antagonistic forms of the mutant G129R (underlined) with remote amino acids 1-14.

Fig illustrates nucleotide and amino acid sequence of growth hormone and its antagonistic forms, mutant G120R (underlined).

Fig is a schematic illustration of a tandem prolactin.

Fig. (A) diagram of the constructs rigid tandem GH with restriction enzymes cut sites, obtained by means of genetic engineering, Notl and Nrul, which makes possible the connection of the linker directly with integral cables with the neighboring domains, and also facilitates changing the linker. (B) Scheme of a hard construct PRL-linker-SN with restriction enzymes cut sites, obtained by means of genetic engineering, Notl and Nrul, which have the same function as in (A), the Notl site is located in the linker sites and, thus, can just be added to a truncated gene RRL in the domain of A. (C) Schematic diagram of rigid tandem PRL, a unique restriction site must be created by genetic engineering, using the degeneracy of the amino acid code, on the border between the linker and PRL in domain B to allow easy synthesis and a modification of this tandem gene.

Materials and methods

Modification of the linker in tandem GH initiated from gene molecules GH(G4S)-GH (χ1C1), which was modified to remove 30 amino acids, the excess N-end of the expressed protein, this gene was also subcloned into a modified vector red(+) receive RET:χ1C1b (Figure 3).

For constructs with helical linkers with flexible ends of the designed linker by ligating to each other complementary oligonucleotides; these oligonucleotides were designed to encode the desired linker and had ends that were luigirules would be in the vector, RET:χ1C1b, which was split Notl and EcoRI. An overview of these linkers is shown in Figure 4.

For rigid linker between domains GH these domains GH had to be modified for hair removal is them-the end (GH1) and N-end (GH2) to these domains ended at the end of the spirals. Then designed restriction sites, using the technique of degenerate codons that would make possible the introduction of new linkers without any interruption of the spiral, which would be formed between the two domains (Figa). Primers were designed to carry out these modifications domains GH GH-tandem (Figb). The resulting construct was called χ15 (6). To generate the linker section, flanked by Notl sites and Nrul, used complementary oligonucleotides were then ligated them into RET:χ15 that splits Notl and Nrul. An overview of these linkers is shown in Fig.7.

The expression constructs is carried out by the initial transformation of the expression vector pet in strain for expression, E. coli BL21(DE3) CodonPlus RIPL. The expression can be performed in a number of different conditions that include different incubation temperature (for example, room temperature, 37°C), different environments (for example, LB, 2YT, 5YT, etc.), a different point of induction (i.e OD600at which induce culture), different concentrations of IPTG (isopropylthio-β-D galactopyranoside) (or other inductor)used for induction culture, and the time at which collect cells after induction.

To the end of this construct, you can add a His-tag, which will facilitate its purification using the-W immobilized affinity chromatography with metal ions (Ni 2+or With2+columns). Constructs that do not have a His-tag, can be cleaned using different methods, such as ion exchange chromatography, hydrophobic column and gel chromatography. To obtain a suitable protein purity may require one or more than one of these cleaning methods.

Design χ15

Modified GH domains was generated using PCR (polymerase chain reaction) and relevant primers. GH1 modified using DiGHNcoGF and GH[AEA3]NotR, and GH2 modified with Ecol-(Nru)GH-F and GHΔ*-HR. The reaction medium PCR consisted of: 1 μl of 100 pmol/ál direct primer, 1 μl of 100 pmol/μl reverse primer, 1 ál pTrcHisGHstop (diluted), 1 μl 10 mm dNTPs, 5 ál of 10x buffer for amplification, 1 µl of 50 mm MgSO4and 0.5 ál of polymerase Pfx, 39,5 ál of sterile water. PCR was performed on these reaction mixtures, using the following temperature profile: 95°C for 5 min; 15×(95°C for 45 s, 55°C for 45 s, 72°C for 45 s; 72°C for 5 min PCR Products were checked using agarose gel, and purified the desired PCR product. Modified GH1 ligated in re:χ1C1b between sites Ncol and Notl obtaining re:χ14. Modified GH2 ligated in re:χ14 between sites EcoRI and HindIII to obtain re:χ15. An overview of this process is shown in Fig.

Change linkers

Phosphorylation of primers

When a gene is the radio linker required 2 or more than 2 duplex primers, primers containing internal 5'-end, was first fosforilirovanii. For each subject to phosphorylation primer was prepared by the following reaction mixture: 2 μl of 100 pmol/μl of oligonucleotide, 2 μl of 10x kinase buffer, 2 μl 10 mm ATP, 13 μl of sterile water, 1 μl T4 polynucleotide kinase (10 units/ál). These components were incubated at 37°C for 30 min and then at 70°C for 10 minutes Then diluted samples 1:10 with buffer for annealing (10 mm TRIS, 50 mm NaCl, 1 mm EDTA (ethylenediaminetetraacetic acid), pH 7.5 to 8.0), with solution of 0.1 pmol/μl. These components could then be used in the reaction annealing described below.

Annealing of the primer duplexes

Primers were diluted to a concentration of 0.1 pmol/μl, using a buffer for annealing (10 mm TRIS, 50 mm NaCl, 1 mm EDTA, pH 7.5 to 8.0). 10 μl of complementary primers were mixed in a fresh tube. This tube is then incubated at 95°C for 2 min and let the temperature fall to 30°C over a period of time 40-60 minutes when it takes more than one duplex primers were mixed with equal volumes of duplexes primers to obtain a solution containing all duplexes primers required for the formation of the desired linker. Then store these solutions on ice.

Ligation and transformation

Approximately 200 ng of the vector, split the relevant restriction is armentani (for example re:χ1C1b, split Notl and EcoRI, or RET:χ15, split Notl and Nrul), incubated with 4 μl of primer after annealing, 1 μl of ligase buffer, 2 μl of T4 DNA ligase and reaction medium was brought to 10 μl with sterile water. These components were incubated overnight in a beaker with ice, which was allowed to melt during this time. 5 ál of the product ligase reaction formed during the night, then added to 50 ál of chemically competent E.coli cells SURE. Incubated them on ice for 1 hour, then exposed to heat shock at 42°C for 30 s was Added to the cells 450 ál of LB medium, and then incubated with the sample for 30 min at 37°C. Then centrifuged mini-culture for 5 min at 4000 rpm, the resulting precipitate resuspendable in 50 μl of LB medium, and then transferred to plates of LB containing carbenicillin (100 μg/ml), tetracycline (10 µg/ml) and glucose (0,3% wt./vol.). Incubated them overnight at 37°C. Resulting colonies were then subjected to screening to check whether the change of the linker successful.

Modification of the General strategy

Generation of constructs with rigid linkers using restriction enzymes Notl and Nrul for splitting RET:χ15, after transformation generated a large number of colonies on plates with the negative control (no duplex primers in the reaction ligation), so was is difficult to implement screening for positive clones. Cleared them by dephosphorylation of the split vector; 15 ál rat:χ15, split Notl and Nrul, was mixed with 2 μl of CIAP buffer 10x, 1 μl of CIAP (alkaline phosphatase calf intestine) (10 units/ál) and 2 ál of sterile water. This mixture is incubated at 37°C for 1 hour and then at 80°C for 30 minutes Then was purified DNA from solution using the cleaning kit (e.g., Qiagen PCR Purification Kit). All the primers used to generate duplexes primers were fosforilirovanii using the method described above. Phosphorylated duplexes primers then ligated in dephosphorylating vector, as described above.

Cloning and expression χ1L1

χ1L1 consists of two domains of the growth hormone GH and subsequent single extracellular domain of the growth hormone, and each of these domains currently associated with the linker (Gly4Ser)4(Fig). The nucleotide sequence χ1L1 shown in Fig.9, and the amino acid sequence shown in Figure 10.

χ1L1 designed by ligating the hGH gene, flanked by sites Nhel and Xhol, χ12 (GHRa-GH-GHRb); this gave χ11. The domain of the GHR then ligated in χ11 between sites EcoRI and HindIII to obtain χ1L1. Schematic diagram of this procedure is shown in 11.

Gene χ1L1 was checked by sequencing and showed that he is correct. Expression was performed using modificirowan the th vector red(+) in E. coli cells BL21 (DE3) CodonPlus RIPL. Protein expressed in LB medium at 4 hours after induction with 1 mm IPTG (final concentration) at OD6000,5-0,6, was partially soluble, and Western band, and analyzed for the presence of GH (Fig), observed bands with different M.M. (molecular weight).

Version χ1L1 with C-terminal His-tag was purified using With2+column (Fig). In the protein preparation has survived a number of contaminating proteins, and Western band was still observed bands with different M.M. Preliminary bioassays of this protein preparation showed that he had a significant agonistic activity (Fig).

Design tandems prolactin and tandems prolactin growth hormone

Constructs tandems PRL (prolactin) and GH were generated using standard techniques of PCR, followed by legirovaniem and transformation of the received vector. The linker can be modified by ligation and transformation oligonucleotide pairs subjected to annealing, the resulting vectors. Below shows three examples of strategies for the design tandem PRL and GH.

Strategy 1

Generation of PRL-(G4S)4-PRL

1. PCR PRL between sites Ncol and Notl (forward primer = atatccatgggcTTGCCCATCTGTCC: reverse primer = atatatatatatgggcggccgccGCAGTTGTTGTTGTGG).

2. Cleavage of the PCR product by restrictase Ncol and Notl.

3. Splitting the recipient vector Ncol and Notl→RET(m)χ1C1b (i.e. GH(G4S) -GH).

4. Ligation of PCR product into a vector with getting pET21(m)PRL(G4S)4-GH

5. PCR PRL between sites EcoRI and HindIIII (forward primer = atatgaattcTTGCCCATCTGTCC; reverse primer = atataagcttGCAGTTGTTGTTGTGG).

6. Cleavage of the PCR product by restrictase EcoRI and HindIII.

7. Splitting the recipient vector EcoRI and HindIII→pET21(m)PRL(G4S)4-GH.

8. Ligation of PCR product into a vector with getting pET21(m)PRL(G4S)4-PRL.

Strategy 2

Generation of PRL-A(EA3K)2A-GH

1. PCR PRL between sites Ncol and Notl (forward primer = atatccatgggcTTGCCCATCTGTCC: reverse primer = atatatatatatgggcggccgccGCAGTTGTTGTTGTGG).

2. Cleavage of the PCR product by restrictase Ncol and Notl.

3. Splitting the recipient vector Ncol and Notl→RET(m)Teak (i.e. SN-A(EA3K)2A-GH).

4. Ligation of PCR product into a vector with obtaining PRL-A(EA3K)2A-GH.

Strategy 3

Generation PPL(EA3K)4A-PRL

1. To anneal the primers to generate a linker And(EA3K)4A.

2. Split the recipient vector restrictase Notl and EcoRI→pET21(m)PRL(G4S)4-PRL (from example 1 above).

3. To ligitamate oligonucleotide dimer in the vector with obtaining PRL(EA3K)4A-PRL.

The above strategy is illustrated in Fig.

EXAMPLE 1

Semi-rigid tandems

Cells of E. coli BL21 (DE3) CodonPlus-RIPL were grown in 10 ml LB medium supplemented with carbenicillin the nom, tetracycline and chloramphenicol. These cells were grown on a shaker at 37°C. the Culture was induced at OD600equal to 0.4 to 0.7 using IPTG at a final concentration of 1 mm. These cultures were grown for an additional 4 h before collection. Cells were literally using a combination of lysozyme, desoxycholate sodium and ultrasonic treatment. The soluble fraction was then isolated by centrifugation. Gels PAGE (polyacrylamide gel electrophoresis), colored Kumasi, did not show clear bands in the expression of the tandem.

The soluble fraction was determined by ELISA (enzyme-linked immunosorbant assay), and 40 ng/well of tandem were loaded on 12%PAGE gel. Protein was transferred to PVDF (polyvinylidene fluoride) membrane and subjected to Western-blotting using rabbit anti-GH antibodies (primary) and anti-rabbit-HRP (conjugated to horseradish peroxidase) antibodies (secondary); see Fig, the Bioactivity of tandem GH containing a semi-rigid linker shown in Figg.

EXAMPLE 2

Hard tandems

Cells of E. coli BL21 (DE3) CodonPlus-RIPL were grown in 10 ml LB medium supplemented with carbenicillin, tetracycline and chloramphenicol. These cells were grown on a shaker at 37°C. the Culture was induced at OD600equal to 0.4 to 0.7 using IPTG at a final concentration of 1 mm. These cultures were grown for an additional the 4 h before collection.

Cells were literally using a combination of lysozyme, desoxycholate sodium and ultrasonic treatment. The soluble fraction was then isolated by centrifugation. Gels PAGE, colored Kumasi, did not show clear bands in the expression of the tandem.

The soluble fraction was determined by ELISA and 40 ng/well of tandem were loaded on 12%PAGE gel. Protein was transferred to PVDF membrane and subjected to Western-blotting using rabbit anti-GH antibodies (primary) and anti-rabbit-HRP antibodies (secondary); see Figg. The bioactivity of tandem GH containing a semi-rigid linker shown in Figg.

EXAMPLE 3

Cleaning tandems

Constructs T1cEAK2+3His and T1cEAK2+4His included in the final list for further study based on their initial spermicidally activities in bioanalysis. Expression of the plasmid was used to transform cells of E. coli BL21 (DE3) CodonPlus-RIPL and spent the expression in 1 l batch cultures. Purification was performed on the soluble protein fraction, using a combination of Ni-chelate affinity chromatography on a column with immobilized metal ions (IMAC), and ion-exchange chromatography. The IMAC was the first purification step, initially, the elution was carried out using a gradient of pH (from pH 8 to pH 3); however, discovered that a large amount of protein was lost in the leaching column. Therefore AB is ora went back to using elution with imidazole (from 0 to 0.5 M imidazole) and with modifications in the cleanup strategy, the authors have achieved more than 70%purity. Then used ion-exchange column (resource Q) to further purify the protein to more than 90%purity. This is illustrated in Fig.

Quantitative assessment based on the results of ELISA: T1cEAK2+3His (RQ13/4)=215 µg/ml; T1cEAK2+3His (RQ14/4)=177 µg/ml.

Each received 1 ml, therefore, gave the common output 392 µg. The receipt was obtained from 2 liters of culture→output per liter=~200 µg.

Activity T1cEAK2+3His reaches over multiple induction than rhGH, at higher concentrations of protein. A similar result can be obtained when the tandem test on a molar basis, see Figb and 18V. A similar analysis was carried out in relation to T1cEAK2+4His. Purification and bioactivity illustrated in Figg, D and 18E.

Quantitative assessment based on the results of the ELISA T1cEAK2+4His (RQ13/4), 550 μg/ml Each received 1 ml, thus giving a total output of 550 µg. The receipt was obtained from 2 liters of culture → output per liter = ~275 mcg. Activity T1cEAK2+4-His reaches over multiple induction than rhGH, at higher concentrations of protein. A similar result can be obtained when the tandem test on a molar basis.

EXAMPLE 4

The concentration χ1C3 was measured using analysis by Bradford (Bradford's). rhGH (~1 mg/ml) was measured in parallel to confirm the validity of the data obtained from analysis by Bradford. Then used χ13 for the nternet replacement standards GH in bioanalysis on GH with obtaining the standard curve of the tandem. Samples of purified and unpurified tandem and rhGH was measured relative to a standard curve GH and standard curve tandem, then measured the protein concentration of each plate ELISA. GH ELISA gives approximately two-thirds of the actual value of tandems when measured through ELISA. This is illustrated in Fig.

EXAMPLE 5

Tandems prolactin/GH

Tandems of prolactin and/or GH with their respective antagonistic mutation or without it, it is possible to synthesize, using PCR to introduce suitable restriction sites at each end of the gene to allow ligation in tandem gene.

Flexible tandems

Tandem gene construct by linking two protein domains with a flexible linker sequence based (G4S)n; at each end of the protein domains and the linker has a unique restriction sites (Fig).

Therefore, two protein domain in this tandem can be modified by ligating in different domains. For example, prolactin (PRL), a mutant prolactin G129R with remote amino acids 1-14 (Δ1-14PRL.G129R), growth hormone (GH) and antagonistic mutant growth hormone G120R (GH.G120R) can be combined in a tandem gene in a variety of ways (Figb). Fig and 23 show the nucleotide sequence and protein sequence for these domains.

To generate gene with the desired protein, the house is nom, flanked suitable sites for restriction endonucleases, you can use the standard PCR. Cleavage of the PCR product and the recipient vector these restriction endonucleases and subsequent legirovaniem and transformation generates tandem with the desired protein domains. This process can be performed on any protein domain or linker (Fig), which would be replaced by using oligonucleotide dimer, as has already been described.

Semi-rigid tandems

Tandem gene construct by linking two protein domains with the helical linker on the basis of sequence And(EA3K)nA; at each end of the protein domains and the linker has a unique restriction sites (Fig).

Therefore, two protein domain in this tandem can be modified by ligating in different domains. For example, prolactin (PRL), a mutant prolactin G129R with remote amino acids 1-14 (Δ1-14PRL.G129R), growth hormone (GH) and antagonistic mutant growth hormone G120R (GH.G120R) can be combined in a tandem gene in a variety of ways (Fig). Fig and 23 show the nucleotide sequence and protein sequence for these domains.

To generate gene with the desired protein domain, flanked appropriate sites of restriction endonucleases, you can use the standard PCR. Cleavage of the PCR product and rezi the inert vector these restriction endonucleases and subsequent legirovaniem and transformation generates tandem with the desired protein domains. This process can be performed on any protein domain or linker (Fig), which would be replaced by using oligonucleotide dimer, as has already been described.

Hard tandems

Two domains of the tandem want to link directly through To the end of α-helix domain and N-terminal α-helix of domain B. Therefore, the genes for these proteins (Fig and 23) in the domain a and B need to be cut so that the helical linker [A(EA3K)nA] was connected directly with these spirals.

Therefore:

ProteinThe truncation of the domain aThe truncated B domain
GH184-1911-6
PRL191-1991-14

Tandem gene construct by linking two protein domains with the helical linker on the basis of sequence And(EA3K)nA; at each end of the protein domains and the linker has a unique restriction sites (Fig). A unique Notl site was constructed in the N-terminal end of the linker section, and a unique Nrul site was developed in the C-terminal end of GH (Figure 5 and 6); it makes possible the modification of the linker section in tandem GH./p>

N-terminal sequence of the linker that includes a Notl site, you can directly add to the PRL gene in the position domain And allowing to construct constructs based on the matrix PRL-linker-GH (Fig). However, in cases where domain B is a PRL (Fig), on the border between the linker and domain B, you must enter a unique restriction site.

Therefore, two protein domain in this tandem can be modified by ligating different truncated domains. For example, prolactin (PRL), a mutant prolactin G129R with remote amino acids 1-14 (Δ1-14PRL.G129R), growth hormone (GH) and antagonistic mutant growth hormone G120R (GH.G120R) can be combined in a tandem gene in a variety of ways (Fig). Depending on their position in the domain a or domain B of protein domains will need to be cut, as described above.

To generate gene with the desired protein domain, flanked appropriate sites of restriction endonucleases, you can use the standard PCR. Cleavage of the PCR product and the recipient vector these restriction endonucleases and subsequent legirovaniem and transformation will generate tandem with the desired protein domains. This process can be performed on any protein domain or the linker, and it is similar to the methodology used for flexible and semi-flexible linkers (Fig).

1. The polyp is Ted, consisting of two binding receptor polypeptide growth hormone, connected in tandem peptide linker molecule, which at least consists of 1-4 copies of the amino acid sequences AND(EAAAK)AND, where specified, the polypeptide is an agonist of the receptor for growth hormone.

2. The polypeptide according to claim 1, where the linker molecule optionally includes at least one flexible non-spiral plot consisting of the amino acid sequence GGGGS and located on its aminoterminal and/or carboxyterminal the end.

3. The polypeptide according to claim 2, where the flexible nonhelical plot localized on aminoterminal the end of the peptide linker molecule.

4. The polypeptide according to claim 2, where the flexible nonhelical plot localized on carboxyterminal the end of the peptide linker molecule.

5. The polypeptide according to claim 2, where the flexible nonhelical plot localized at the amino - and carboxyterminal ends of the peptide linker molecule.

6. The polypeptide according to claim 1, where the specified peptide linker connects the carboxyl end of the first polypeptide growth hormone with amino end of polypeptide growth hormone.

7. The polypeptide according to claim 6, where the peptide linker is located between the last amino acid residue C-terminal helix of the first polypeptide growth hormone and the first amino acid residue N-terminal helix of storagerelated growth hormone.

8. Nucleic acid encoding a polypeptide with properties of the receptor agonist of growth hormone, which has a nucleotide sequence that determines the amino acid sequence of the polypeptide according to claim 1.

9. The vector for expression of the polypeptide with properties of the receptor agonist of growth hormone, comprising the nucleic acid of claim 8.

10. The selected cell, transformed or transfusiona vector according to claim 9, which produces a polypeptide with properties of the receptor agonist of growth hormone.



 

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19 cl, 11 ex, 7 tbl

FIELD: biotechnology, molecular biology, proteins.

SUBSTANCE: invention relates to a method for preparing cytokines of class II and can be used in medicine. Prepared proteins zcyto20, zcyto22, zcyto24 and zcyto25 are the most relative with interferon-α at amino acid sequence level. Receptor of cytokines of class II represents a receptor for this family of proteins. Proteins can be prepared by recombinant way using a cell-host transformed with expression vector that comprises nucleic acids corresponding to proteins. Base on proteins xcyto20, xcyto21, zcyto22, zcyto24 and zcyto25 antiviral pharmaceutical composition and specific antibodies are prepared. Invention provides preparing the novel cytokine that stimulates cells of differentiation hemopoietic line and possesses the expressed antiviral activity.

EFFECT: valuable biological and medicinal properties of polypeptide, improved preparing method.

24 cl, 21 tbl, 32 ex

FIELD: gene engineering, in particular method for treatment of viral infections.

SUBSTANCE: protein ZCYTO21 has amino acid sequence which is nearly similar to amino acid sequence of interferon-α. Protein and antibodies thereto have antiviral activity and are useful in treatment of hepatitis B and C as well as other diseases.

EFFECT: new protein with antiviral activity.

71 cl, 1 dwg, 6 tbl, 7 ex

FIELD: biotechnology, medicine.

SUBSTANCE: Zalpha 11-ligand is isolated from cDNA library generated from activated cells of human peripheral blood that have been selected from CD3. Animal is inoculated with Zalpha 11-ligand and antibodies are prepared that are able to bind specifically with epitopes, peptides or polypeptides of Zalpha 11-ligand. Invention provides effective regulation and/or development of hemopoietic cells in vitro and in vivo. Invention can be used for preparing Zalpha 11-ligand and antibodies for it.

EFFECT: valuable properties of new cytokine.

18 cl, 5 tbl, 1 dwg, 55 ex

The invention relates to the field of biotechnology, specifically to obtain protein/factor inhibiting osteoclastogenesis (OCIF), and can be used for treatment and immunological diagnosis of diseases involving bone resorption

The invention relates to the field of genetic engineering and can be used in the biomedical industry

The invention relates to a protein characterized by the following properties: (a) molecular weight during electrophoresis in polyacrylamide gel in the presence of LTOs (SDS-PAGE), which constitutes approximately 60 KD in terms of recovery, approximately 60 KD and 120 KD in non conditions; (b) high affinity for the cation-exchange column and a column of heparin; (b) biological activity of inhibiting the differentiation and/or maturation of osteoclasts, and this activity is reduced by heating at 70oC for 10 min or 56oC for 30 min, the activity is lost when heated at 90oC for 10 min; (g) internal amino acid sequences presented in SEQ ID NO: 1, 2, and 3, and (d) with optional N-terminal amino acid sequence represented in SEQ ID NO: 7; a method for production of such proteins by culturing human fibroblasts; protein purification by a combination of ion-exchange column chromatography, affinity column chromatography and column chromatography with reversed phase

The invention relates to the field of the biotechnology industry, in particular the production of virus-inducer for the production of human leukocyte interferon

FIELD: biotechnologies.

SUBSTANCE: recombinant DNA is produced, which codes functionally active hybrid protein (BrdGl7ACA-cbd), consisting of amino-acid sequence of acylase glutaryl-7- aminocephalosporanic acid of strain Brevundimonas diminuta All-Russian collection of industrial microorganisms B-1297 and chitin-binding domain of chitinase Al Bacillus circulans. Recombinant plasmid pSVH0108 is constructed for expression of BrdGl7ACA-cbd in cells E.coli, containing sequence of recombinant DNA that codes hybrid protein under control of promotor and terminator of RNA-polymerase of phage T7. As a result of E.coli strain tranformation with this recombinant plasmid and selection of transformed clones, a new strain E.coli BL21(DE3)/pSVH0108 cbd is produced - producer of hybrid protein BrdGl7ACA-cbd.

EFFECT: high yield of recombinant ferment.

4 dwg, 2 tbl, 7 ex

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